Real-time analysis of preprotachykinin promoter activity in single cortical neurons

Technological limitations have hindered the study of gene elements regulati ng transcription within CNS neurons. In the present studies, rat cortical b rain slices endogenously expressing the preprotachykinin (PPT) gene were tr ansfected with gene constructs encompassing green fluorescent protein (GFP) under the control of the PPT promoter. These slices were maintained in org anotypic culture so that the fluorescence intensity within individual livin g cells could be quantified using laser scanning confocal microscopy before and after application of stimulatory agents. Combined treatment with forsk olin and elevated potassium significantly increased expression of both endo genous PPT mRNA and the PPT promoter-GFP construct. The ability to follow f luorescence changes within single neurons in real time offers a powerful "w ithin-subject" experimental approach for analysis of neural gene promoters.